TBX6相关性先天性脊柱侧凸临床表型及致病机制研究

发布时间：2018-09-17 08:21

【摘要】：研究背景：先天性脊柱侧凸(Congenital scoliosis, CS)是由胚胎期椎体发育异常导致的脊柱侧方弯曲≥10°。CS具有进展快、畸形重、并发症多等特点,严重时可致患者瘫痪,是造成青少年残疾的主要疾病之一,给家庭和社会带来极大负担。脊椎动物的脊柱是由体前中胚层周期性形成的体节发育而来,这一过程叫做体节形成(somitogenesis),体节形成异常即可导致先天性椎体畸形。由于其确切病因不明,目前尚无有效的预测手段,治疗仍以支具或手术治疗来缓解病情发展为主,因此探寻其发病原因,对于从病因学领域控制发病率、早期诊断和早期干预这种严重致残性脊柱畸形疾病、缓解家庭和社会负担具有重要意义。CS可由基因异常或环境因素变化导致,随着越来越多的遗传学病因报道,遗传因素在CS发病中起到的作用逐渐被人们重视。CS是一种复杂、多基因、多遗传模式疾病。目前国际上对于CS的遗传学研究主要集中于单基因或染色体水平改变致病的家系研究或病例报道,而在点突变和染色体突变之间存在大片空白,即拷贝数变异(Copy number variation, CNV) 。在前期研究中,我们采用Agilent的人类全基因组CGH芯片对20例CS散发病例进行全基因组扫描,在两个病例(10%)的16p11.2区域都发现了一个同样大小de novo缺失。该区域的TBX6基因在人类体节形成中起重要作用,TBX6基因突变或者缺失会产生脊柱发育障碍等表型。研究目的：研究TBX6相关性CS的致病率、致病机制及临床表型,并建立相应动物模型。研究方法：首先通过qPCR、靶向CGH芯片及TBX6基因全长测序对237例散发CS患者16p11.2缺失及TBX6基因突变进行检测,之后对筛选出的TBX6相关性CS与非TBX6相关性CS进行表型分析及对比,明确TBX6相关性CS表型特征。进一步通过CRISPR-Cas9构建TBX6相关性CS的斑马鱼模型,验证其表型特征及致病机制。研究结果：在237名散发CS患者中共发现23例TBX6无效变异,包括17例16p11.2/TBX6缺失,5例TBX6基因移码突变及1例TBX6基因无义突变,并证明rs2289292-rs3809624-rs3809627单倍型T-C-A是除TBX6无效变异外CS发病的必要条件。表型分析证实TBX6相关性CS患者均存在一个或一个以上节段半椎体/楔形椎畸形,椎体畸形以半椎体/楔形椎为主(82.4%),TBX6相关性CS主要累及节段为下胸段及上腰段(T7-L3,82.2%),平均累累及椎体数为2.61,倾向于累及更少的脊柱节段；在CS合并肋骨畸形比例上,TBX6相关性与非TBX6相关性CS间不存在显著差异,但有更高比例的简单型肋骨畸形(60.9%)；此外,TBX6相关性CS倾向于更低的椎管内畸形发生率(8.7%)。在斑马鱼模型中,tbx6及tbx16根据不同程度的突变表现出呈梯度的椎体畸形。研究结论：16p11.2/TBX6缺失是散发CS的重要遗传学病因,其致病机制可能为TBX6剂量不足。该病因能解释约10%的CS。我们将16p11.2缺失/TBX6无效变异合并T-C-A亚效等位基因的先天性脊柱侧凸定义为TBX6相关性先天性脊柱侧凸(TBX6-associated congenital scoliosis),其在临床表型上也具有自身特点。在斑马鱼模型中,致病机制与表型均符合TBX6相关性CS的剂量模型,但其具体机制仍待进一步研究。
[Abstract]:BACKGROUND: Congenital scoliosis (CS) is a lateral curvature of the spine (> 10 degrees) caused by abnormal vertebral development during embryonic period. CS is characterized by rapid progress, severe deformities and multiple complications. It can cause paralysis in severe cases. It is one of the main diseases causing disability in adolescents and brings great burden to family and society. The spine of an animal develops from somatogenesis, a process called somatogenesis, which is periodically formed in the anterior mesoderm. Abnormal somatogenesis can lead to congenital vertebral deformities. It is of great significance for controlling the incidence of CS from the field of etiology, early diagnosis and early intervention to relieve the burden of family and society. CS is a complex, multi-gene, multi-genetic model disease. At present, the genetics of CS is mainly focused on single gene or chromosome level change pathogenic family studies or case reports, but there is a large gap between point mutation and chromosome mutation, namely Copy number variation. In the previous study, we used Agilent's human genome-wide CGH chip to scan the entire genome of 20 sporadic CS cases. In both cases (10%) of the 16p11.2 region, an identical deletion of de novo was found. The TBX6 gene in this region plays an important role in the formation of human somatic segments, and the TBX6 gene mutates or deletes. Objective: To study the pathogenicity, pathogenesis and clinical phenotype of TBX6-related CS, and to establish animal models. Methods: TBX6 gene mutation and 16p11.2 deletion were detected in 237 sporadic CS patients by qPCR, CGH chip and TBX6 gene full-length sequencing. TBX6-related CS and non-TBX6-related CS were selected for phenotypic analysis and comparison. TBX6-related CS phenotypic characteristics were clarified. A zebrafish model of TBX6-related CS was constructed by CRISPR-Cas9 to verify its phenotypic characteristics and pathogenic mechanism. 2/TBX6 deletion, 5 TBX6 gene shift mutation and 1 TBX6 gene nonsense mutation proved that rs2289292-rs3809624-rs3809627 haplotype T-C-A was a necessary condition for the onset of CS except for the invalid variant of TBX6. Phenotypic analysis confirmed that one or more segmental hemivertebra/wedge vertebral malformations were present in all TBX6-related CS patients. TBX6-related CS mainly involved lower thoracic and upper lumbar segments (T7-L3,82.2%). The average number of vertebrae involved was 2.61, which tended to involve fewer vertebral segments. There was no significant difference between TBX6-related CS and non-TBX6-related CS in the ratio of CS with rib deformity, but there was a higher proportion of simple rib deformity (60%). In zebrafish models, Tbx6 and tbx16 exhibit gradient vertebral malformations based on varying degrees of mutation. The study concludes that 16p11.2/TBX6 deletion is an important genetic cause of sporadic CS, and its pathogenesis may be due to the inadequate dose of TBX6. TBX6-associated congenital scoliosis (TBX6-associated congenital scoliosis) is defined as a congenital scoliosis associated with the deletion of 16p11.2/TBX6 invalid variant and T-C-A subtype. It also has its own characteristics in clinical phenotype. In zebrafish models, the pathogenesis and phenotype are consistent with TBX6-related CS. However, the specific mechanism remains to be further studied.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R687.3

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